WO2013084772A1 - 細胞の測定方法及び細胞の測定用試薬 - Google Patents

細胞の測定方法及び細胞の測定用試薬 Download PDF

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WO2013084772A1
WO2013084772A1 PCT/JP2012/080760 JP2012080760W WO2013084772A1 WO 2013084772 A1 WO2013084772 A1 WO 2013084772A1 JP 2012080760 W JP2012080760 W JP 2012080760W WO 2013084772 A1 WO2013084772 A1 WO 2013084772A1
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atp
methanol
sample
cells
alanine
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PCT/JP2012/080760
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English (en)
French (fr)
Japanese (ja)
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雅弘 岡野定
野田　英之
福薗　真一
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株式会社日立ハイテクノロジーズ
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Priority to EP12855701.4A priority Critical patent/EP2789692B1/de
Priority to US14/362,645 priority patent/US9290789B2/en
Publication of WO2013084772A1 publication Critical patent/WO2013084772A1/ja

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence

Definitions

  • the present invention relates to a method and a reagent for measuring the number of cells or the presence of cells with high sensitivity by measuring intracellular ATP.
  • the present invention relates to methods and reagents for measuring cells (particularly live cells) in a sample.
  • Cell count measurement methods are used in a wide range of fields such as pharmaceutical, medical, food, brewing, and water treatment.
  • fields such as pharmaceutical, medical, food, brewing, and water treatment.
  • the presence or absence of cells in the sample and the number of cells are measured for sample quality control.
  • bacterial substances bacteria / fungi
  • it is essential to manage bacterial substances (bacteria / fungi) contained in pharmaceutical raw materials, intermediates, final products, and pharmaceutical water based on the Japanese Pharmacopoeia established by the Ministry of Health, Labor and Welfare. Body count is being carried out.
  • the main method for measuring the number of bacteria and fungi prescribed by the Japanese Pharmacopoeia is the culture method.
  • a sample is brought into contact with an agar plate medium and cultured, and one bacterium forms one colony by culturing, and the number of colonies (CFU: Colony Forming Unit) is calculated. Quantify as a number.
  • the problem of the culture method in the pharmaceutical industry is that the culture time is about one week. For this reason, it is necessary to wait for the measurement result at the stage of production of the intermediate or shipment of the final product, and the culture method is a time and economical burden for the business operator.
  • the type of the medium must be selected according to the bacterial species. For example, a standard agar medium for general aerobic bacteria, an R2A medium for bacteria that prefer an oligotrophic environment, a thioglycol medium for anaerobic bacteria, a PDA medium for fungi, etc. Must be selected (Non-Patent Document 1).
  • aerobic bacteria are a standard agar medium at a temperature of 30 to 35 ° C for 48 to 72 hours
  • anaerobic bacteria are a thioglycolic acid medium at a temperature of 30 to 35 ° C for 5 days or more
  • bacteria that prefer an oligotrophic environment are A culture medium suitable for colony formation of each bacterial species, such as culturing in a R2A medium at a temperature of 20-25 ° C (or 30-35 ° C) for 4-7 days, and a fungus in a PDA medium at a temperature of 20-25 ° C for 5 days or more. Multiple protocols such as temperature and incubation time are required.
  • ATP Addenosine TriPhosphate, adenosine triphosphate
  • luminescence measurement method is known as a rapid measurement method for the number of cells with reduced measurement time.
  • ATP to be measured is an organic compound that all living organisms have in their cells and that is an energy source required for the vital activities of the cells.
  • the ATP luminescence measurement method uses luciferase and luciferin that chemically react with ATP to generate light, measures the luminescence produced by the reaction of intracellular ATP, luciferase, and luciferin, and estimates the number of cells from the amount of luminescence. is there.
  • ATP from live cells or dead cells, or sample 102 containing free ATP ATP from live cells or dead cells, or sample 102 containing free ATP, (1) removal of ATP from living cells 103, (2) Extraction of ATP in living cells 104, (3) Three steps of luminescent reaction and luminescence measurement 105 with ATP from living cells and luminescent reagents (luciferase, luciferin, etc.), and ATP and luminescence in living cells From the luminescence with the reagent, the number of viable cells is estimated.
  • luminescent reaction and luminescence measurement 105 with ATP from living cells and luminescent reagents (luciferase, luciferin, etc.), and ATP and luminescence in living cells From the luminescence with the reagent, the number of viable cells is estimated.
  • the ATP measurement method 101 in the living cell of the conventional method the live cell 122 in the ATP luminescence method 121, the ATP derived from the dead cell, the free state ATP 123, and the state in each step of the ATP degrading enzyme 124 will be described.
  • ATP-degrading enzyme 124 such as apyrase is added to the sample (106). Since dead cells have broken cell walls or cell membranes, ATP and free ATP ⁇ 123 in dead cells are degraded by activity 125 of ATP-degrading enzyme 124 (126). On the other hand, since the living cell 122 is alive 127, ATP in the living cell is isolated from the ATP-degrading enzyme 124 by the cell wall or cell membrane of the living cell (Patent Document 2).
  • ATP in living cells can be increased by adding glucose to Escherichia coli, Lactobacillus brevis, Staphyllococcus aureus, and Bacillus subtilis after culturing in a standard medium.
  • germination is induced by adding glucose and alanine to spores.
  • ATP extraction step 104 in the living cell “destruction of the cell wall of the living cell” and “inactivation of the ATP-degrading enzyme” are performed.
  • ATP extract 107 of trichloroacetic acid, benzalkonium chloride, and methanol is added (Patent Document 2).
  • the cell wall of the living cell is destroyed by the additive, whereby the living cell is killed and ATP is extracted (128).
  • the ATP-degrading enzyme loses its activity due to the enzyme-denaturing action of the additive (129), even if ATP is extracted from living cells, ATP is not degraded and is preserved.
  • the extracted ATP derived from living cells and the luminescent reagent 108 are brought into contact with each other to generate a luminescent reaction 130.
  • the generated luminescence is quantified using a luminescence measuring device such as a luminometer. Since the amount of luminescence is proportional to the number of ATP derived from living cells, and the number of ATP is proportional to the number of living cells, the number of living cells can be estimated from the amount of luminescence. Note that these three steps (live extracellular ATP removal step 103, intracellular ATP extraction step 104, and ATP luminescence reaction / measurement step 105) proceed irreversibly.
  • the ATP measurement sensitivity of a general ATP luminescence measurement method is 1 x 10 -15 to 1 x 10 -16 mol (1 to 0.1 fmol). So far, the inventor has achieved an ATP measurement sensitivity of 1.0 ⁇ 10 ⁇ 18 mol and has attempted to measure one living cell.
  • this ATP luminescence measurement method when used in the pharmaceutical manufacturing field, especially in the management of pharmaceutical water, it detects cells contained in highly pure water such as purified water and water for injection, in other words, water that contains almost no nutrients. In such a case, there is a problem that one cell of viable bacteria cannot be measured by the conventional technique.
  • Patent Document 2 shows an increase in ATP due to amino acids, alanine is not exemplified, and the ATP increasing action of alanine is not recognized. Further, since Patent Document 3 does not show any ATP increasing action of glucose or alanine, it is extremely difficult to predict the ATP increasing action of alanine from these two known examples.
  • the conventional technology and a combination of these conventional technologies can be used for various types of cells such as bacteria, fungi (yeasts and molds), spores and spores, non-spores, aerobic and anaerobic, gram negative and gram positive. It was insufficient to measure simultaneously with two ATP luminescence measuring methods.
  • an object of the present invention is to provide a method and means capable of simultaneously measuring a plurality of types of cells (particularly living cells) with high sensitivity by the ATP luminescence measurement method.
  • the present inventor has found that methanol contributes to an increase in ATP in living cells (live bacteria) in the ATP luminescence measurement method, and methanol is added before the ATP extraction step. As a result, it was found that a plurality of types of cells (particularly living cells) can be measured simultaneously and with high sensitivity, and the present invention has been completed. That is, the present invention includes the following [1] to [15].
  • a first step of increasing ATP in living cells by adding methanol to a sample suspected of containing living cells A method for measuring cells in a sample, comprising a second step of extracting intracellular ATP, and a third step of causing the extracted ATP to emit light.
  • the cells are captured on the filter by filtration through a filter, and after removing the solution containing methanol, the second step and the third step are performed. [1] to [3] The method described.
  • the genus Bacillus, Pseudomonas, Methylobacterium, Escherichia, Staphylococcus, Clostridium The method according to any one of [1] to [10], wherein at least one microbial cell selected from the group consisting of the genus (Clostridium), the genus Candida, and the genus Aspergillus is used.
  • Bacillus subtilis Pseudomonas aeruginosa, Pseudomonas fluorescence, Methylobacterium extorquens, Escherichia coli (Escherichia coli), Staphylococcus aureus, Clostridium sporogenes, Candida albicans, and at least one fungus selected from the group consisting of Aspergillus niger The method according to any one of [1] to [10], which is used.
  • the present invention provides a method and a reagent for simultaneously and highly sensitively measuring a plurality of types of cells (particularly living cells) in a sample based on the ATP luminescence measurement method.
  • bacteria, fungi yeast, mold
  • spores and spores non-spores
  • aerobic and anaerobic, gram-negative and gram-positive cells can be measured with one measurement method. It is possible to measure the amount of ATP inclusion exceeding 1 amol / CFU. Even when the number of cells in the sample is small or the amount of ATP in the cells is small, the cells can be detected and the number of cells can be measured.
  • the present invention can be applied to various pharmaceutical manufacturing fields, cosmetics manufacturing fields, clinical medicine, basic biochemistry, and the like, and is particularly effective for detecting cells and fungus bodies in an oligotrophic environment such as management of pharmaceutical water.
  • FIG. 4 is a diagram showing the state of live cells, dead cell ATP, free ATP, and ATP-degrading enzyme in the ATP luminescence measurement method, compared with the steps of the present invention and the conventional method in the intracellular ATP luminescence measurement method. It is a figure which shows the residual activity of the ATP degradation enzyme with respect to methanol concentration. It is a figure which shows the viability of B.subtilis, S.aureus, and C.albicans with respect to methanol concentration. It is a figure which shows the effect of various additives with respect to the amount of ATP inclusion of M.extorquens. It is a figure which shows the amount of ATP inclusion of M.extorquens with respect to methanol concentration.
  • the present invention relates to a method for measuring cells in a sample based on the ATP luminescence measurement method.
  • a plurality of types of cells for example, bacterial cells
  • methanol and also glucose, alanine
  • ATP-degrading enzyme 145 is added.
  • the “increasing ATP in living cells” step 150 and the “removing ATP outside living cells” step 143 are performed simultaneously.
  • the feature of the present invention is that it has been found that methanol contributes to an increase in ATP in living cells (viable bacteria). This was originally found by the present inventors who are not shown in Patent Document 2 and Patent Document 3.
  • methanol is used in the conventional technology used in the “extraction of ATP in cells” step 144.
  • methanol is used in the ATP extraction step, and the cell wall of the cell is used. The cells (cells) are destroyed by destruction, and ATP is extracted from the cells (Patent Document 4).
  • methanol is used before the “extraction of intracellular ATP” step 144 to increase the amount of ATP in the cell under the condition that the cell is alive,
  • the effect obtained is completely different from that of methanol used in the ATP extraction process (for example, Example 3).
  • Methanol has an enzyme-denaturing action to denature proteins such as enzymes, but the present inventor can use methanol at a concentration that retains the activity of ATP-degrading enzyme and a concentration that allows living cells to survive. was also confirmed (Examples 1 and 2). Since methanol has an enzyme-denaturing action as described above, if necessary, luminescence measurement may be performed by removing methanol prior to the “extraction of intracellular ATP” step 144.
  • methanol having a final concentration of 0.01 to 30% (v / v)%, glucose, alanine, and ATP-degrading enzyme 145 are brought into contact with a sample 142 containing plural types of live cells, dead cell ATP, and free ATP.
  • a sample 142 containing plural types of live cells, dead cell ATP, and free ATP.
  • heating is performed at 25 to 50 ° C. for about 5 to 360 minutes in order to increase the ATP in the live cell and decompose the ATP outside the live cell.
  • methanol, glucose, alanine and ATP-degrading enzyme may be removed by filtration (146) with a filter.
  • ATP extract 147 is added to destroy the cell wall of the cell, extract ATP from the cell (144), and inactivate the ATP-degrading enzyme.
  • the extracted ATP is brought into contact with the luminescent reagent 148 to cause a luminescence reaction 149, and the amount of luminescence can be quantified to quantify the number of living cells in the sample.
  • a first step in which methanol is added to a sample suspected of containing living cells to increase ATP in the living cells, A second step of extracting intracellular ATP and a third step of emitting the extracted ATP are performed.
  • the sample is not particularly limited as long as it is a sample suspected of containing living cells to be measured, and may be a liquid, solid or gas sample, or a mixed sample thereof.
  • the liquid sample can be used as it is, or diluted or concentrated with a solvent.
  • the solid sample may be suspended in a solvent, homogenized by a pulverizer or the like, or a supernatant obtained by stirring with a solvent may be used.
  • a stick such as a cotton swab may be moistened with sterile water, and the measurement location may be wiped off, then rinsed in a solvent, and the resulting liquid used as a sample.
  • the solvent include distilled water, physiological saline, phosphate buffer, Tris buffer, and the like.
  • samples include food and beverage production, clinical examination (urine, feces, blood, etc.), pharmaceutical production, cosmetic production, wastewater treatment, bacterial examination (seawater, river water, industrial water, soil, etc.), antibacterial properties, etc. Includes any sample in testing, cleanliness testing, etc.
  • a sample can be appropriately prepared by those skilled in the art using methods and means conventionally used in the ATP luminescence measurement method.
  • the cell to be a measurement control is not particularly limited as long as it is a cell containing ATP.
  • the object of the present invention is to measure whether or not cells are contained in the sample to be measured, but the contained cells are unknown and difficult to specify. Therefore, when using this measurement method, it is desirable to measure an unknown sample after confirming that this measurement method is normal.
  • Bacillus genus Bacillus genus, Pseudomonas genus, Methylobacterium genus, Escherichia genus, Staphylococcus genus, Clostridium genus, Candida (Clostridium genus) It is preferable to use at least one cell selected from the group consisting of the genus Candida and the genus Aspergillus.
  • Bacteria Gram-positive bacteria such as aerobic spore bacteria Bacillus subtilis, aerobic bacteria Staphylococcus aureus, aerobic Listeria monocytogenes, anaerobic spore bacteria Of Clostridium sporogenes; Gram-negative bacteria such as aerobic bacteria Pseudomonas aeruginosa, Pseudomonas fluorescence, Methylobacterium extorquens, Escherichia coli, anaerobic Salmonella (Salmonella), Legionella (Legionella); Fungi: yeasts such as Candida albicans, Saccharomyces cerevisiae; Aspergillus niger.
  • nine types of living cells defined by the Japanese Pharmacopoeia (B. subtilis, Pseudomonas aeruginosa, fluorescent bacteria, Methylobacterium extrusens, E. coli, Staphylococcus aureus, Clostridium sporogenes, Candida albicans, And a positive control comprising 2 or more, more preferably 5 or more, and most preferably all 9 of Aspergillus niger).
  • methanol is added to a sample suspected of containing live cells.
  • concentration of methanol added is less than 50% final concentration, preferably 0.1-30%, more preferably 0.1-5%, for example 1%.
  • a filter can be immersed in a methanol solution, or a methanol solution can be dripped or sprayed on a filter.
  • a substance selected from sugar and alanine is added to the sample.
  • sugar at least one selected from the group consisting of glucose, fructose and sucrose can be used.
  • Sugar is added so that the final concentration is 0.8 ⁇ m or less, preferably 0.1 ⁇ m or less.
  • Alanine is added so that the final concentration is 0.5 ⁇ mM or less, preferably 0.1 ⁇ mM or less.
  • the final concentration of methanol is 1%
  • the final concentration of sugar is 0.1 ⁇ mM
  • the final concentration of alanine is 0.1 ⁇ mM.
  • ATP-degrading enzyme is added simultaneously with methanol.
  • ATP-degrading enzymes are known in the art, and known ATP-degrading enzymes can be used alone or in combination under appropriate reaction conditions (time, temperature and pH). Specific examples include ATP-degrading enzymes described in Patent Document 1 (Patent No. 3547882), such as adenosine phosphate deaminase, apyrase, alkaline phosphatase, acid phosphatase, hexokinase, and adenosine triphosphatase.
  • Kits for carrying out the ATP luminescence measurement method are commercially available, and the ATP-degrading enzyme attached to the kit can be used.
  • a filter when using a filter as a sample, a filter can be immersed in an ATP decomposing enzyme solution, or an ATP decomposing enzyme solution can be dripped or sprayed on a filter.
  • methanol (optionally sugar and alanine) may be added to the sample, and the reaction may be performed by adding ATP-degrading enzyme to the sample simultaneously with or after the addition, or ATP-degrading enzyme may be added to the sample, At the same time or after the addition, methanol (optionally further sugar and alanine) may be added to the sample to carry out the reaction.
  • ATP degrading enzyme may be added, or ATP degrading enzyme is added and the live extracellular ATP removing step is performed, Methanol may be added after removing the ATP-degrading enzyme therein. From the viewpoint of ease of operation, it is preferable to carry out the reaction by sequentially adding methanol and an ATP-degrading enzyme to the sample.
  • the reaction between the sample and methanol and / or ATP-degrading enzyme varies depending on the type of ATP-degrading enzyme used, the concentration of methanol, the type of cells to be measured, etc., but 25 to 50 ° C., preferably 30 to 45
  • the reaction is carried out at 1 ° C. for 1 minute or longer, preferably 1 minute to 6 hours, more preferably 5 minutes to 2 hours.
  • ATP degrading enzyme it is preferable to remove methanol from the sample after the live cell ATP increasing step (first step). Therefore, for example, after the above step, it is preferable to perform the second step and the third step after capturing the cells in the sample by filtration with a filter and removing the solution containing methanol. Moreover, it is preferable to remove or inactivate the ATP degrading enzyme after the live extracellular ATP removal step.
  • the removal of ATP-degrading enzyme can be performed, for example, by filtration with a filter, addition of an inhibitor of ATP-degrading enzyme, or the like.
  • an ATP extract having an activity of inactivating ATP-degrading enzyme may be used.
  • the first step and the second step can be performed on the filter using the filter as a sample.
  • the removal of the solution containing methanol and the removal of the ATP-degrading enzyme can be easily performed by washing the filter with pure water or a buffer solution.
  • intracellular ATP is extracted.
  • a known method in the ATP luminescence measurement method for example, a method in which an ATP extract is added to a sample and intracellular ATP is extracted outside the cell can be used.
  • the ATP extract is not limited as long as it can extract ATP from the inside of the cell, and an ATP extract that can inactivate ATP-degrading enzyme is also known.
  • Such ATP extracts are known and are commercially available. For example, trichloroacetic acid (TCA), a surfactant (benzalkonium chloride, benzethonium chloride, etc.), lysozyme and the like can be mentioned.
  • a filter can be immersed in an ATP extract, or an ATP extract can be dripped or sprayed on a filter.
  • the extracted ATP is caused to emit light.
  • a method generally employed in the ATP luminescence measurement method for example, luciferin-luciferase luminescence reaction can be used.
  • the luciferin-luciferase luminescence reaction is performed by adding a luminescence reagent containing luciferin and luciferase to the sample. Since the luminescence reagent reacts with the extracted ATP to generate luminescence, the amount of luminescence is measured.
  • the luminescent reagent either natural luciferase or mutant luciferase may be used, and a commercially available product can be used.
  • the ATP extraction sample may be collected from the filter, and a luminescence reagent may be added to the ATP extraction sample to emit light, or the filter may be immersed in the luminescence reagent or be attached to the filter. A luminescent reagent may be dropped to emit light on the filter.
  • Luminescence can be measured by a luminescence measurement method known in the art, for example, using a luminometer, a luminescence plate reader, or by photon counting using a photomultiplier tube. By measuring the amount of luminescence, the cells in the sample can be measured.
  • “measuring cells” means detecting the presence of cells in a sample and measuring the number of cells. According to the method of the present invention, it is possible to measure cells in a sample, particularly a plurality of types of cells (live cells) in a single method, with high sensitivity, for example, with an ATP inclusion amount exceeding 1 amol / CFU. .
  • the reagent of the present invention is characterized by containing methanol.
  • the reagent of the present invention may further contain a sugar (for example, at least one selected from the group consisting of glucose, fructose and sucrose), and alanine.
  • the reagent of the present invention may be supplied together with at least one reagent selected from the group consisting of an ATP-degrading enzyme, an ATP extract, and a luminescent reagent, and these reagents are preferably packaged together.
  • the form of the reagent can be any form such as a solution, a powder, or a granule.
  • each component is included in a form and concentration suitable for carrying out the method of the invention.
  • the reagent of the present invention may further include instructions for carrying out the method for measuring cells.
  • a bacterial suspension of M.extorquens was prepared according to the following procedure.
  • M.extorquens was shake-cultured at 25 ° C. for 72 hours in 5 ⁇ mL of SCD liquid medium (Nissui Pharmaceutical).
  • the culture solution (medium) was dispensed into a tube and centrifuged at 10,000 rpm for 10 minutes to collect the bacteria at the bottom of the tube.
  • the supernatant was removed, water was added instead to suspend the bacteria, and the mixture was centrifuged again at 10,000 rpm for 10 minutes to collect the bacteria at the bottom of the tube. This process was repeated three times to prepare a bacterial suspension excluding the medium components.
  • the suspension was cultured in water for injection for 3 days at 37 ° C. to obtain a bacterial suspension for measuring ATP luminescence.
  • the Lucifer HS set (Kikkoman Biochemifa) was used as a reagent for ATP luminescence measurement.
  • the Lucifer HS set includes an ATP-erasing enzyme, an ATP extraction reagent, and a luminescence reagent.
  • ATP increase / decrease effect of glucose and alanine on B. subtilis and M. extorquens was evaluated according to the following procedure. Glucose (Wako Pure Chemical) 10 ⁇ L, alanine (Wako Pure Chemical) 10 ⁇ L, and ATP-degrading enzyme 10 ⁇ L were added to B. subtilis and M. extorquens bacterial suspension 50 ⁇ L. Finally, phosphate buffer (pH 7.4) (Invitrogen) was added to adjust the total volume to 100 ⁇ L.
  • the final concentrations of glucose, alanine, and ATP-degrading enzyme were set to 100 MmM, 50 mM, and 10 (v / v)%, respectively, and each mixture was heated at 37 ° C for 30 minutes.
  • addition of glucose and alanine was excluded as a protocol described in the reagent set instructions.
  • M.extorquens could not be measured with high sensitivity, and it was difficult to simultaneously measure B. subtilis and M.extorquens with a single ATP luminescence measurement method. .
  • M. extorquens is a methanol-assimilating bacterium, in the examples described later, the effect of increasing ATP by methanol was examined.
  • Table 2 shows the amount of luminescence of the ATP / luciferase / luciferin reaction with respect to the methanol concentration.
  • Table 2 shows the amount of luminescence generated by mixing an ATP solution containing 0.1 to 50 (v / v)% methanol (sample No. 2 to 5), an ATP solution not containing methanol (sample No. 1), and a luminescent reagent. Shows a comparison.
  • the emission amounts of 12703 CPS (Count per second), 10200 CPS, and 11534 CPS were 88-110%, respectively.
  • the emission ratio decreases to 29%
  • the concentration of methanol mixed in the ATP luminescence reaction / measurement process is preferably 1% (v / v)% or less.
  • Example 1 the concentration of methanol capable of maintaining the activity of ATP-degrading enzyme was examined.
  • ATP erasing solution containing ATP-degrading enzyme (ATP erasing reagent attached to Kikkoman Biochemifa's Lucifer HS Set) and 80 ⁇ L of methanol with adjusted concentration were mixed.
  • ATP solution 2 ⁇ 10 6 amol / 10 ⁇ L was added thereto, and the mixture was allowed to stand at 37 ° C. for 30 minutes.
  • the final concentration of methanol was adjusted to 0.1 to 80 (v / v)%.
  • the mixture was diluted with water to a methanol concentration of 0.1 (v / v)% or less that does not affect the ATP luminescence reaction (Reference Example 2, Table 2). . Subsequently, 50 ⁇ L of this diluted solution (1 ⁇ 10 3 amol ATP / 50 ⁇ L) was added to 50 ⁇ L of the luminescence reagent, and the amount of luminescence was measured. A 1 ⁇ 10 3 amol / 50 ⁇ L ATP solution not added with ATP-degrading enzyme was prepared, and the amount of luminescence with respect to this ATP solution was also measured.
  • the amount of luminescence of the ATP solution without ATP-degrading enzyme added is considered to be almost the same as the amount of luminescence of the ATP solution when ATP is added after the activity of ATP-degrading enzyme is lost. It was defined as 0%.
  • Fig. 2 shows the residual activity and methanol concentration of ATP-degrading enzyme.
  • the ATP luminescence reaction was not measured from the mixture of ATP-degrading enzyme and ATP solution in the presence of 0 to 50% (v / v)% methanol. This indicates that the ATP-degrading enzyme retained the activity in the concentration range of 0 to 50% (v / v)% methanol and decomposed the added ATP.
  • the methanol concentration exceeded 50% (v / v)%, the activity of the ATP-degrading enzyme began to decrease, and when the methanol concentration was 80% (v / v)%, the enzyme activity decreased to 20%.
  • Example 2 In this example, since methanol originally has a bactericidal effect, the influence of the methanol concentration on the evaluation bacteria was examined.
  • Fig. 3 shows the viability of bacteria for each methanol concentration.
  • B. subtilis showed a survival rate of 100% even when exposed to 80% (v / v)% methanol.
  • S. aureus and C. albicans showed a survival rate of 90% or more with methanol of 30% (v / v)% or less.
  • Example 3 In this example, the ATP increase effect of methanol on M. extorquens was examined.
  • a bacterial suspension of M.extorquens was prepared according to the following procedure.
  • M.extorquens was shake-cultured at 25 ° C. for 72 hours in 5 ⁇ mL of SCD liquid medium (Nissui Pharmaceutical).
  • the culture solution (medium) was dispensed into a tube and centrifuged at 10,000 rpm for 10 minutes to collect the bacteria at the bottom of the tube.
  • the supernatant was removed, water was added instead to suspend the bacteria, and the mixture was centrifuged again at 10,000 rpm for 10 minutes to collect the bacteria at the bottom of the tube. This process was repeated three times to prepare a bacterial suspension excluding the medium components.
  • the suspension was cultured in water for injection for 3 days at 37 ° C. to obtain a bacterial suspension for measuring ATP luminescence.
  • each bacterial sample is diluted with water, and then the bacterial suspension is applied to R2A agar medium and cultured at 25 ° C. After 96 hours from the start of culture, 168 hours later The number of colonies was counted.
  • the first embodiment using the prepared bacterial suspension is shown.
  • the first form is luminescence measurement only by mixing various reagents.
  • Glucose (Wako Pure Chemical Industries, Ltd.), Alanine (Wako Pure Chemical Industries, Ltd.), ATP-degrading enzyme, 10% methanol, and phosphate buffer (pH 7.4) (Invitrogen Corporation) are added to 50 ⁇ L of the M.extorquens suspension. The total volume was adjusted to 100 ⁇ L, and when glucose, alanine, and methanol were not added, the total volume was adjusted to 100 ⁇ L with phosphate buffer (Table 3, Sample Nos. 1 to 5). After the mixture was heated at 37 ° C. for 30 minutes, 100 ⁇ L of the ATP extract was added to 100 ⁇ L of the mixture in the live extracellular ATP removal / intracellular ATP increase step (Table 3, Sample Nos. 1 to 4).
  • the second form employs filtration, and filtration is used for the purpose of collecting bacteria and removing methanol.
  • M.extorquens bacterial suspension 50 ⁇ L was subjected to centrifugal filtration (2000 rpm) for 1 minute using Ultrafree MC (Millipore, pore size 0.45 ⁇ m, polyvinylidene fluoride membrane filter), and the bacteria were collected on the filter.
  • 10 ⁇ L each of glucose, alanine, ATP-degrading enzyme and 10% methanol and 70 ⁇ L of phosphate buffer solution (pH 7.4) were added onto the filter to prepare a total mixture of 100 ⁇ L.
  • the final concentrations of glucose and alanine were 0.1 ⁇ mM, and the final concentrations of ATP-degrading enzyme and methanol were 10% and 1%, respectively (Table 3, Sample No. 6).
  • the mixture on the filter was heated at 37 ° C. for 30 minutes. After the heating, in order to remove the mixed solution containing methanol, all of the mixed solution was subjected to centrifugal filtration with Ultra Free MC (2000 rpm for 1 minute), and the mixed solution as the filtrate was discarded. Subsequently, 200 ⁇ L of water was added onto the filter and filtered to wash the filter. The washing was repeated 5 times. After washing, 100 ⁇ L of ATP extract was added onto the filter, allowed to stand for 5 minutes, and then subjected to centrifugal filtration (2000 rpm, 1 minute) to collect the filtrate. 50 ⁇ L of the sample after each ATP extraction was added to 50 ⁇ L of the luminescence reagent, and the generated luminescence was measured.
  • Table 3 shows the amount of ATP inclusion (amol / CFU) per CFU of M. extorquens in the intracellular ATP luminescence measurement method.
  • Sample No. 1 and Sample No. 2 are the same results as Example 1.
  • the amount of ATP in M.extorquens increased to 0.9 amol / CFU, avoiding inhibition and about less than the conventional method (Comparative Example 1 of Reference Example 1).
  • Example 4 In this example, the relationship between the amount of ATP and the amount of glucose and alanine in M. extorquens was examined.
  • a bacterial suspension of M.extorquens was prepared according to the following procedure.
  • M.extorquens was shake-cultured at 25 ° C. for 72 hours in 5 ⁇ mL of SCD liquid medium (Nissui Pharmaceutical).
  • the culture solution (medium) was dispensed into a tube and centrifuged at 10,000 rpm for 10 minutes to collect the bacteria at the bottom of the tube.
  • the supernatant was removed, water was added instead to suspend the bacteria, and the mixture was centrifuged again at 10,000 rpm for 10 minutes to collect the bacteria at the bottom of the tube. This process was repeated three times to prepare a bacterial suspension excluding the medium components.
  • the suspension was cultured in water for injection for 3 days at 37 ° C. to obtain a bacterial suspension for measuring ATP luminescence.
  • Figure 4A shows the amount of ATP encapsulated M.extorquens per 1 CFU (amol / CFU) when the alanine concentration was changed from 0.1 to 50 ⁇ mM in the presence of 1% (v / v)% methanol and 100 ⁇ mM glucose. It is. The amount of ATP encapsulated increased from an alanine concentration of 10 mM or less, and 0.9 amol / CFU at 1 mM or less.
  • Fig. 4B shows the amount of ATP encapsulated per 1 CFU (amol / CFU) when the glucose concentration was changed from 0.1 to 100 mM in the presence of 1% (v / v)% methanol and 50 mM mM alanine.
  • the amount of ATP encapsulated remained at about 0.5 ⁇ amol / CFU even when the glucose concentration was changed.
  • FIG. 4C shows the amount of ATP encapsulated per 1 CFU when the concentrations of glucose and alanine are simultaneously changed to 0.1 mM, 5 mM, and 50 mM in the presence or absence of methanol.
  • the amount of ATP encapsulated increased rapidly from less than 5 mM in the absence of methanol, and showed 0.9 amol / CFU at 0.1 mM. This result is the same as the result of the combination of 0.1 mM alanine, 100 mM glucose and 1% methanol (Fig. 4A). However, when 1% methanol was added to 0.1 mM alanine and 0.1 mM glucose, the amount of ATP encapsulated increased to 1.2 amol / CFU. This indicates that the amount of ATP is increased by adding alanine and glucose to less than 5 mM and adding methanol.
  • Patent Document 3 Japanese Patent Application Laid-Open No. 2006-174751 shows that glucose and alanine are effective in promoting germination of spore bacteria, but does not show any ATP increasing effect. As the result of FIG. 4, the present inventors have found that there is an optimum concentration range for the effect of increasing ATP, depending on each or a combination of glucose and alanine.
  • a bacterial suspension of M.extorquens was prepared according to the following procedure.
  • M.extorquens was shake-cultured at 25 ° C. for 72 hours in 5 ⁇ mL of SCD liquid medium (Nissui Pharmaceutical).
  • the culture solution (medium) was dispensed into a tube and centrifuged at 10,000 rpm for 10 minutes to collect the bacteria at the bottom of the tube.
  • the supernatant was removed, water was added instead to suspend the bacteria, and the mixture was centrifuged again at 10,000 rpm for 10 minutes to collect the bacteria at the bottom of the tube. This process was repeated three times to prepare a bacterial suspension excluding the medium components.
  • the suspension was cultured in water for injection for 3 days at 37 ° C. to obtain a bacterial suspension for measuring ATP luminescence.
  • Fig. 5A shows the amount of ATP encapsulated in M.extorquens with respect to 0-10% methanol concentration.
  • the amount of ATP encapsulated in M. extorquens showed the maximum value (1.5 amol / CFU) at a methanol concentration of 0.5%, and decreased by 60% (0.7 amol / CFU) at a methanol concentration of 0%.
  • the ATP inclusion amount 1.1 ⁇ amol / CFU
  • the amount of ATP encapsulated decreased by 90% (0.15 amol / CFU) (Fig. 5B).
  • Example 6 According to the Japanese Pharmacopoeia, the following nine types of bacteria are defined as the cells used in the medium test to confirm that the medium used for “pharmaceutical water management” and “sterility testing” is appropriate.
  • aerobic spore bacteria gram positive) Bacillus subtilis (ATCC 6633), aerobic bacteria (gram negative) Pseudomonas aeruginosa (NBRC 13275), aerobic bacteria (gram negative) Pseudomonas fluorescence (NBRC 15842), aerobic bacteria ( Gram negative) Methylobacterium extorquens (NBRC 15911), Aerobic bacteria (Gram negative) Escherichia coli (ATCC 11775), Aerobic bacteria (Gram positive) Staphylococcus aureus (ATCC 6538), Anaerobic spore bacteria (Gram positive) Clostridium sporogenes (ATCC) 11437), fungus (yeast) Candida albicans (ATCC 10231), fungus (m
  • intracellular ATP measurement was performed on nine species of indicator bacteria described in the Japanese Pharmacopoeia.
  • a bacterial suspension of non-spore-forming bacteria (P.aeruginosa, P.fluorescence, M.extorquens, S.aureus, E.coli, C.Calbicans) was prepared according to the following procedure. Each bacterium was added to 5 mL of SCD liquid medium and cultured with shaking for 48 hours. The culture temperature was 37 ° C for S. aureus and E. coli, and 25 ° C for P.fluorescence, P.aeruginosa, M.extorquens, and C.albicans, based on the Japanese Pharmacopoeia. The culture solution after the culture was dispensed into a tube, and centrifuged at 10,000 rpm for 10 minutes to collect the bacteria at the bottom of the tube.
  • the supernatant containing each component was removed, and water was added instead to suspend, and the bacteria were collected again by centrifugation at 10,000 rpm for 10 minutes. This process was repeated 3 times to remove each component and replace with water. Subsequently, the suspension was cultured in water for injection for 3 days at 37 ° C. to obtain a bacterial suspension for measuring ATP luminescence.
  • Table 4 shows the ATP increase effect of methanol, glucose and alanine on the above-mentioned 9 official bacterial species in the ATP luminescence measurement method.
  • the present invention relates to a plurality of types of bacteria such as bacteria and fungi (yeasts and molds), spores and spores, non-spores, aerobic and anaerobic, gram-negative and gram-positive, It is possible to detect with high sensitivity.
  • this invention is not limited to the above-mentioned Example, Various modifications are included.
  • the above-described embodiments have been described in detail for easy understanding of the present invention, and are not necessarily limited to those having all the configurations described.
  • a part of the configuration of one embodiment can be replaced with the configuration of another embodiment, and the configuration of another embodiment can be added to the configuration of one embodiment.

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WO2016147313A1 (ja) * 2015-03-17 2016-09-22 株式会社日立製作所 薬剤感受性試験装置及び薬剤感受性試験キット並びに薬剤感受性試験方法
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CA3069500A1 (en) 2017-07-12 2019-01-17 Ecolab Usa Inc. A method for the rapid detection of bacterial spores in an industrial process
CN110272968A (zh) * 2019-03-15 2019-09-24 李文杰 ATP荧光lgCB-lgIB标曲法评价一次性卫生用品真菌杀灭效果的方法
CN110272963A (zh) * 2019-03-15 2019-09-24 李文杰 ATP生物荧光lgCA-lgIA标准曲线法评价日化品真菌杀灭效果的方法
JP2021052675A (ja) * 2019-09-30 2021-04-08 高砂熱学工業株式会社 除染効果の判定方法

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JPWO2016147313A1 (ja) * 2015-03-17 2017-06-22 株式会社日立製作所 薬剤感受性試験装置及び薬剤感受性試験キット並びに薬剤感受性試験方法
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EP3608418A1 (de) 2018-08-06 2020-02-12 Hitachi, Ltd. Mikroorganismustest

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